Assays for detecting the presence or amount of an anti-drug antibody

ABSTRACT

Methods and kits for detecting antibodies (e.g., anti-drug antibodies). Such methods and kits permit the detection of, for example, anti-drug antibodies in human body fluids, such as blood, plasma and serum.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/619,422, filed Feb. 11, 2015, now U.S. Pat. No. 9,759,732, whichclaims the benefit of U.S. Provisional Patent Application Ser. No.61/938,556, filed Feb. 11, 2014, the entire contents of which are herebyincorporated by reference.

TECHNICAL FIELD

This invention relates to methods and kits for detecting the presence ofanti-drug antibodies in a sample, and more particularly to methods andkits for detecting anti-drug antibodies in the presence of a drug in thesample.

BACKGROUND

The introduction of biotherapeutics (e.g., biologic agents such asproteins, peptides, nucleotides, etc.) has given a major boost to thetreatment of diseases such as inflammatory bowel disease, ankylosingspondylitis, multiple sclerosis and rheumatoid arthritis. In many casesthese biological agents have proven very successful in clinicalpractice. Biologic agents, including therapeutic antibodies, are knownto have immunogenic potential, and administration of therapeuticproteins to a patient can induce immune response leading to theformation of anti-drug antibodies (“ADAs”). Such ADAs may reduce theeffectiveness of the therapeutic protein. For example, they may bind toor/and neutralize the therapeutic protein, resulting in changes of drugpharmacokinetics or pharmacodynamics that alters drug efficacy. ADAs maycause serious side effects, including allergic reactions,cross-reactivity against endogenous proteins by neutralizing antibodies(NAbs), and complement activation. The production of ADAs have beendescribed for several monoclonal antibodies available for the treatmentof rheumatoid arthritis (adalimumab and infliximab), Crohn's disease(infliximab), multiple sclerosis (natalizumab and alemtuzumab) andplaque psoriasis (adalimumab). In some patients, the clinical benefitsprovided by such therapeutic proteins diminishes over time due to theformation of ADAs. Immungenicity risk assessment is critical tounderstand frequency and severity for drug induced ADA. NAbcross-reactive to endogenous protein causing depletion syndrome has beenreported (erythropoietin).

With an increasing number of therapeutic proteins approved for clinicaluse, the immunogenicity of such products has become informative toclinicians, manufacturers and regulatory agencies. It iswell-established that certain substances will affect the detection orquantitation of an analyte in immunoassays (or ligand binding assays).These interference factors including but not limited to circulatingdrugs negatively impact assay specificity, accuracy, and sensitivity.“Drug interference” that reduce ADA assay “drug tolerance” is regardedas a major technical challenge for immunogenicity assessment to monitorADA as part of patient's monitoring for drug clinical safety andefficacy.

Although the above approaches demonstrated some improvement in drugtolerance, sensitivity and relative accuracy is not maintained incomparison to no-drug ADA detection therefore risking false negative andunder-reporting ADA incidence and titers in treated patients. Despiteindustry regulatory guidance documents and white papers recommendingsensitivity between 250 and 500 ng/mL [Shankar G, Devanarayan V,Amaravadi L et al.: Recommendations for the validation of immunoassaysused for detection of host antibodies against biotechnology products.Journal of Pharmaceutical and Biomedical Analysis 48(5), 1267-1281(2008); Mire-Sluis A R, Barrett Y C, Devanarayan V et al.:Recommendations for the design and optimization of immunoassays used inthe detection of host antibodies against biotechnology products. Journalof Immunological Methods 289(1-2), 1-16 (2004)], drug tolerance issometimes evaluated without any acceptance criteria and clinicalprotocols are then written instructing long wash-out periods beforeantibody measurement to allow for drug clearance and the avoidance offalse negative results due to drug interference. This approach is notdesired due to risks in missing ADA assessment in early time pointsespecially in the case with a long half-life drug and/or multi-dosingregimen and the wash out period approach is not feasible. Somenon-ligand binding based methods such as mass spectrometry has beenevaluated for PK in the presence of ADA interference, the expected assaysensitivity has not been acceptable and enrichment of analyte is neededwhich is ligand binding based which poses the similar challenges.

A variety of assay formats have been used with success to detect ADAs,including ELISA (direct, indirect and bridging), radioimmunoassays,electrochemiluminescence, and surface plasmon resonance. The developmentof such assays, however, is often complicated by interference caused bythe presence of the drug. The challenge of analytical interferences inligand binding assays has long been recognized. With the advent oflong-lived monoclonal antibody therapies, the need for specifictechniques to detect ADA in the presence of drug is of particularconcern. The most widely adopted approaches in use currently still havelimitations of timing, sensitivity or accuracy. Thus, there is a need inthe art for methods and kits to more accurately and reproducibly detectthe presence of ADA in samples, such as biological samples.

SUMMARY

The present invention is based, at least in part, on the discovery of anovel assay method that is effective for reducing or eliminating theproblems caused by interference by drug or target in ADA detection. Inparticular, the present invention is based on the development of a novelADA assay comprising the following exemplary steps. First, excess drugmaterial is added to the samples containing potential ADAs (both freeADA and ADA/drug complex) to bind all remaining free ADAs, formingdrug/ADA complexes. Second, these complexes are precipitated usingpolyethylene glycol. Third, after a series of washes to remove serumprotein and immuoglobulin, the final precipitate (drug/ADA complexes) isreconstituted with a solution to dissociate the complexes and thencoated on a large surface (under conditions to keep drug and ADA apart)or substrate (e.g., a high bind carbon plate with high coating capacity)for a time sufficient to allow coating of all dissociated free drugs andfree ADAs. Fourth, specific detection of the total ADA levels is thenperformed using labeled drug. Accordingly, the present invention relatesto methods, compositions and kits for determining the presence or amountof an ADA in a sample (e.g., a biologic sample).

In one embodiment, the final precipitate (drug/ADA complexes) isreconstituted with an acid solution to dissociate the complexes and thencoated on a large surface (under acidic conditions to keep drug and ADAapart) or substrate (e.g., a high bind carbon plate with high coatingcapacity) for a time sufficient to allow coating of all dissociated freedrugs and free ADAs. The acidic environment prevents the complexes fromreforming while being immobilized onto the substrate surface. When anacid solution is used to dissociate the complexes, the assay can bereferred to as a PandA (PEG and Acid) assay.

In another embodiment, the final precipitate (drug/ADA complexes) isreconstituted with a basic solution to dissociate the complexes and thencoated on a large surface (under basic conditions to keep drug and ADAapart) or substrate (e.g., a high bind carbon plate with high coatingcapacity) for a time sufficient to allow coating of all dissociated freedrugs and free ADAs. The basic environment prevents the complexes fromreforming while being immobilized onto the substrate surface.

The selection of an acidic or basic solution will depend on theparameters of the drug (e.g., the biologic drug), such as pI, or thepresence of certain conjugating bonds, and the selection will haveminimal effect on the integrity and structure of the drug.

In some embodiments, the incubation step following the initial acid orbase addition can be carried out at 22° C., 23° C., 25° C., 27° C., 30°C., 32° C., 35° C., 37° C., 39° C. or higher.

In some embodiments, following the final precipitation step, each samplecan be further diluted to a final sample dilution of, e.g., 1:20, 1:25,1:30, 1:40, 1:50, or 1:60.

In one aspect, the disclosure provides a method for determining thepresence or absence of an ADA in a sample, the method comprisingcontacting the sample with an excess amount of drug to which the ADAbinds to form drug/ADA complexes, contacting the drug/ADA complexes withpolyethylene glycol (PEG), to form a precipitate comprising drug/ADAcomplexes, contacting the precipitate with a solution to dissociatingthe drug/ADA complexes, immobilizing the dissociated ADAs on a surfaceand/or substrate, and determining the presence of or amount of said ADA.In a further aspect, the determining step comprises contacting theimmobilized ADA with drug conjugated with a detectable label, anddetermining the presence of or amount of said detectable label, tothereby determine the presence or amount (titer) of ADA in the sample.

In some embodiments, the method for determining the presence or absenceof an ADA in a sample further comprises before, after or part of thedetermining step, determining the amount of ADA in the sample.

In some embodiments, the method for determining the presence or absenceof an ADA in a biological sample further comprises, after theimmobilizing step, treating (e.g., washing) the support to removeunbound drug.

In other embodiments, the method for determining the presence or absenceof an ADA in a biological sample further comprises, after contacting thesample with PEG, washing the precipitate.

In still other embodiments, the method further comprises immobilizingthe drug on the substrate before, after or during the step ofimmobilizing the ADA on the substrate.

In another aspect, the disclosure provides a method for reducinginterference in a drug assay (e.g., a drug PK assay, a drug quantitationassay, or a drug potency assay) due to the presence of an ADA in asample, the method comprising contacting the sample with an excessamount of ADA to saturate free drug and form drug/ADA complexes,contacting the drug/ADA complexes with polyethylene glycol (PEG), tothereby form a precipitate comprising drug/ADA complexes, contacting theprecipitate with a solution to dissociating the drug/ADA complexes,immobilizing the dissociated drug on a substrate under acidicconditions, and performing the drug assay using specific detectionreagent for drug, to thereby reduce interference from the ADA. The drugassay can be, for example, a drug quantitation assay, a drug PK assay ora drug potency assay.

In some embodiments, the method for reducing interference in a drugassay due to the presence of an ADA further comprises determining thepresence or absence of, or the amount of the drug in the sample using ananti-idiotype antibody labeled with a detectable label.

In some embodiments, the methods disclosed herein further comprisediluting the sample before it is contacted with an excess amount ofdrug. For example, the sample is diluted 1:2, 1:5, 1:10, 1:20 foldbefore it is contacted with an excess amount of drug.

In other embodiments, the sample comprises a biological sample, whereinthe biological sample comprises a material selected from the groupconsisting of body fluids, mucus secretions, saliva, blood, whole blood,plasma or serum. In some embodiments, the sample comprises a drug.

In still other embodiments, the drug comprises an antibody or fragmentthereof, a dual affinity antibody, diabody, multiple domain biologics(such as antibody drug conjugate), a nucleic acid (siRNA, antisenseoligonucleotide, gene therapy drugs), a peptide or a polypeptide (nativeor modified), a peptidomimetic, a carbohydrate, a lipid, or an organicor inorganic small molecule compound, or any combinations thereof. Insome embodiments, the drug comprises a therapeutic antibody, a proteintherapeutic, an enzyme, an engineered binding protein, an engineeredantibody-like protein, a fusion protein, a scaffold protein, or anycombinations thereof. When the drug comprises an antibody or fragmentthereof, the antibody may be a murine, human, humanized or chimericantibody. In some embodiments, the drug is a drug modified to exhibitless immunogenicity as compared to the same drug in unmodified form(i.e., the drug has been modified to be less immunogenic).

In some embodiments, the substrate comprises a carbon surface, glasssurface, silica surface, metal surface, a polymeric material, a surfacecontaining a metallic or chemical coating, a membrane, a bead (e.g., amicro-bead), a porous polymer matrix, a substrate comprising cellulosicfibers, or any combinations thereof. The substrate can comprise apolymeric material, wherein the polymeric material is selected from thegroup consisting of polystyrene, polyvinyl chloride, polypropylene,polyethylene, polyamide, and polycarbonate.

In some embodiments, the substrate comprises a porous carbon surface. Inone embodiment, the substrate is a high bind carbon plate.

In some embodiments, the substrate comprises a large surface with highcoating capacity. A substrate comprising a large surface with highcoating capacity includes, for example, a high bind carbon plate (e.g.,a MSD (Meso Scale Discovery®, Rockville Md.).

In some embodiments, the methods provided comprise contacting drug/ADAcomplexes with polyethylene glycol (PEG) to form a precipitatecomprising drug/ADA complexes. The PEG comprises at least one PEGcompound having a molecular weight between 1,000 and 40,000 daltons,including, for example at least one PEG compound selected from the groupconsisting of PEG1000, PEG1450, PEG3000, PEG6000, PEG8000, PEG10000,PEG14000, PEG15000, PEG20000, PEG250000, PEG30000, PEG35000, andPEG40000.

In one or more embodiments, the sample is contacted with PEG at aconcentration of between about 0.1% and about 10.0%, about 0.2% andabout 7.0%, between about 0.5% and about 6.0%, between about 0.5% andabout 5.0%, between about 1.5% and about 5.5%, between about 2.0% andabout 5.0%, between about 3.0% and about 4.5%, between about 3.5% andabout 4.0%, between about 1.0% and about 2.5%, between about 1.2% andabout 1.5%, or about 0.1%, 0.2%, 0.5%, 1.0%, 1.2%, 1.5%, 2.0%, 2.5%,3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%,9.0%, 9.5% or about 10.0% PEG.

In other embodiments, the methods comprise contacting a precipitatecomprising an ADA and/or ADA/drug complex with an acid solution. Theacid solution can comprise an organic acid, an inorganic acid, or amixture thereof. In some aspects, the acid solution comprises an acidselected from the group consisting of citric acid, isocitric acid,glutamic acid, acetic acid, lactic acid, formic acid, oxalic acid, uricacid, trifluoroacetic acid, benzene sulfonic acid, aminomethanesulfonicacid, camphor-10-sulfonic acid, chloroacetic acid, bromoacetic acid,iodoacetic acid, propanoic acid, butanoic acid, glyceric acid, succinicacid, malic acid, aspartic acid, hydrochloric acid, nitric acid,phosphoric acid, sulfuric acid, boric acid, hydrofluoric acid,hydrobromic acid and any combinations thereof. In an exemplaryembodiment, the acid solution comprises acetic acid. For methodscomprising contacting a precipitate comprising an ADA and/or ADA/drugcomplex with an acid solution, the precipitate is contacted with an acidat a concentration of between about 0.1M to about 5M.

In other embodiments, the methods comprise contacting a precipitatecomprising an ADA and/or ADA/drug complex with a base solution. The basesolution can comprise an organic base, an inorganic base, or a mixturethereof. In some aspects, the base solution comprises a base selectedfrom the group consisting of urea, sodium hydroxide, rubidium hydroxide,cesium hydroxide, calcium hydroxide, strontium hydroxide, bariumhydroxide, zinc hydroxide, lithium hydroxide, acetone, methylamine, andammonia. For methods that include contacting a precipitate comprising anADA and/or ADA/drug complex with a basic solution, the precipitate iscontacted with a base at a concentration of between about 0.1 M to about1 M.

In some aspects, the disclosure provides antibodies, anti-drugantibodies and drug labeled with (i.e., conjugated to) a detectablelabel. The detectable label comprises a label selected from the groupconsisting of a hapten, radioactive isotope, an enzyme, a fluorescentlabel, a chemiluminescent label, and electro-chemiluminescent label, afirst member of a binding pair, and a substrate for an enzymaticdetection reaction. In one embodiment, the detectable label comprises anelectrochemiluminescent label comprising a sulfo-TAG® label.

In some embodiments, the detectable label comprises a fluorophore,wherein the fluorophore is selected from the group consisting greenfluorescent protein, blue fluorescent protein, red fluorescent protein,fluorescein, fluorescein 5-isothiocyanate (FITC), cyanine dyes (Cy3,Cy3.5, Cy5, Cy5.5, Cy7), Bodipy dyes (Invitrogen) and/or Alexa Fluordyes (Invitrogen), dansyl, Dansyl Chloride (DNS-C1),5-(iodoacetamida)fluorescein (5-IAF,6-acryloyl-2-dimethylaminonaphthalene (acrylodan),7-nitrobenzo-2-oxa-1,3-diazol-4-yl chloride (NBD-Cl), ethidium bromide,Lucifer Yellow, rhodamine dyes (5-carboxyrhodamine 6G hydrochloride,Lissamine rhodamine B sulfonyl chloride, rhodamine-B-isothiocyanate(RITC (rhodamine-B-isothiocyanate), rhodamine 800); tetramethylrhodamine5-(and 6-)isothiocyanate (TRITC)), Texas Red™, sulfonyl chloride,naphthalamine sulfonic acids including but not limited to1-anilinonaphthalene-8-sulfonic acid (ANS) and6-(p-toluidinyl)naphthalen-e-2-sulfonic acid (TNS), Anthroyl fatty acid,DPH, Parinaric acid, TMA-DPH, Fluorenyl fatty acid,Fluorescein-phosphatidylethanolamine, Texasred-phosphatidylethanolamine, Pyrenyl-phophatidylcholine,Fluorenyl-phosphotidylcholine, Merocyanine 540, Naphtyl Styryl,3,3′dipropylthiadicarbocyanine (diS-C3-(5)), 4-(p-dipentylaminostyryl)-1-methylpyridinium (di-5-ASP), Cy-3 lodo Acetamide,Cy-5-N-Hydroxysuccinimide, Cy-7-Isothiocyanate, IR-125, Thiazole Orange,Azure B, Nile Blue, Al Phthalocyanine, Oxaxine1,4′,6-diamidino-2-phenylindole. (DAPI), Hoechst 33342, TOTO, AcridineOrange, Ethidium Homodimer, N(ethoxycarbonylmethyl)-6-methoxyquinolinium(MQAE), Fura-2, Calcium Green, Carboxy SNARF-6, BAPTA, coumarin,phytofiuors and Coronene.

In some embodiments, the detectable label comprises an enzyme thatcatalyzes a color change reaction, including, an enzyme selected fromthe group consisting of alkaline phosphatase, beta-galactosidase, horseradish peroxidase, urease and beta-lactamase and glucose oxidase.

In some embodiments, the detectable label comprises a first member of abinding pair or a second member of a binding pair, wherein the bindingpair is selected from the group consisting of biotin/streptavidin,biotin/avidin, biotin/neutravidin, biotin/captavidin, epitope/antibody,protein A/immunoglobulin, protein G/immunoglobulin, proteinL/immunoglobulin, GST/glutathione, His-tag/Nickel, antigen/antibody,FLAG/M1 antibody, maltose binding protein/maltose, calmodulin bindingprotein/calmodulin, enzyme-enzyme substrate, and receptor-ligand bindingpairs.

In some embodiments, the detectable label comprises a first member of abinding pair; and the second member of the binding pair is conjugated toan enzyme, an antibody epitope, an antigen, a fluorophore, aradioisotope, a nanoparticle, a member of a second binding pair, and ametal chelate.

In other embodiments, the detectable label comprises a first member of abinding pair, wherein the first member of the binding pair is biotin andthe second member of the binding pair is selected from the groupconsisting of streptavidin, avidin, neutravidin and capravidin, and thesecond member of the binding pair conjugated to an enzyme.

The methods provided herein can be performed on either a manual orautomated instrument platform, depending on the number of samples to betested.

“Anti-drug antibodies” or “ADAs” are antibodies that bind specificallyto any region of a drug. For example, an anti-drug antibody may be anantibody or fragment thereof, which may be directed against any regionof a drug antibody, e.g., the variable domain, the constant domains, orthe glycostructure of the antibody). Such anti-drug antibodies may occurduring drug therapy as an immunogenic reaction of a patient. An ADA maybe one of any human immunoglobulin isotype (e.g., IgM, IgE, IgA, IgG,IgD) or IgG subclass (IgG1, 2, 3, and 4). ADAs include ADAs from anyanimal source, including, for example, human or non-human animal (e.g.veterinary) sources.

For the purpose of the present specification, the term “NAb” or“neutralizing antibody” refers to an antibody that binds to anendogenously produced molecule, e.g., an antibody, nucleic acid,peptide, polypeptide, peptidomimetic, carbohydrate or lipid. Forexample, a NAb may be an endogenously produced protein, such as, forexample, erythropoietin or insulin. The NAb may or may not reduce (e.g.,neutralizes) at least one biological activity of the endogenouslyproduced molecule.

For instance, in some aspects, the disclosure provides a method fordetermining the presence or absence of NAb in a sample, the methodcomprising contacting the sample with an excess amount of antigen towhich the NAb binds to form an antigen/Nab complexes, contacting theantigen/NAb complexes with polyethylene glycol (PEG), to form aprecipitate comprising antigen/NAb complexes, contacting the precipitatewith a solution to dissociate the antigen/NAb complexes, immobilizingthe dissociated NAbs on a surface and/or substrate, and determining thepresence of or amount of said NAb. In a further aspect, the determiningstep comprises contacting the immobilized NAb with of an antigen towhich the Nab binds conjugated with a detectable label, and determiningthe presence of or amount of said detectable label, to thereby determinethe presence or amount (titer) of NAb in the sample.

In the context of the invention, the term “patient” refers to anysubject, preferably a mammal, and more preferably a human, with adisease or suspected of having a disease. The term “subject,” as usedherein, refers to any animal (e.g., a human or non-human animalsubject). In some instances, the subject is a mammal. In some instances,the term “subject”, as used herein, refers to a human (e.g., a man, awoman, or a child). In some instances, the term “subject”, as usedherein, refers to laboratory animal of an animal model study.

As used herein, the term “biological sample” or “sample” refers to asample obtained or derived from a patient which comprises patientderived immunoglobulin and may therefore be referred to as animmunoglobulin sample. By way of example, a biological sample comprisesa material selected from the group consisting of body fluids, blood,whole blood, plasma, serum, mucus secretions, saliva, cerebrospinalfluid (CSF), bronchioalveolar lavage fluid (BALF), fluids of the eye(e.g., vitreous fluid, aqueous humor), lymph fluid, lymph node tissue,spleen tissue, bone marrow, and an immunoglobulin enriched fractionderived from one or more of these tissues. In some embodiments thesample is, or comprises blood serum or is an immunoglobulin enrichedfraction derived from blood serum or blood. The sample is, or can bederived (obtained) from, a bodily fluid or body tissue. In someembodiments, the sample is obtained from a subject who has been exposedto the drug, such as repeatedly exposed to the same drug. In otherembodiments, the sample is obtained from a subject who has not recentlybeen exposed to the drug, or obtained from the subject prior to theplanned administration of the drug.

The term “substrate”, as used herein refers to any material ormacromolecular complex to which an ADA or drug material (e.g., anantibody, nucleic acid, peptide, polypeptide, peptidomimetic,carbohydrate, lipid, or an organic or inorganic small molecule compound)may bind. The composition and/or surface of the substrate should allowfor binding of an ADA or drug material under acidic conditions (or basicconditions) that allow for dissociation of the ADA/drug complexes. Insome embodiments, these substrates have a high loading capacity, whichimproves sensitivity, thus allowing for detection of ADAs and/or drugmaterials present in relatively low concentrations. Examples of commonlyused substrates include, but are not limited to, carbon surfaces (e.g. aporous or high bind carbon plate), glass surfaces, silica surfaces,plastic surfaces, metal surfaces, surfaces containing a metallic orchemical coating, membranes (e.g., nylon, polysulfone, silica),micro-beads (e.g., latex, polystyrene, or other polymer), porous polymermatrices (e.g., polyacrylamide gel, polysaccharide, polymethacrylate),and substrates comprising cellulosic fibers (e.g., cellulose sponges,cellulose paper). In one aspect, the porous or high bind carbon plate isa MSD (Meso Scale Discovery®) high bind plate. The substrate may be abiosensor chip, microarray, or lab-on-chip capable of sensing a targetmolecule. Any kind of biosensor that is capable of sensing specificbinding to the biosensor chip is applicable, including commerciallyavailable biosensors, such as the biosensors produced by Biacore.

As used herein, an entity (e.g., antibody, anti-drug antibody, drug,protein, enzyme, antibody, antibody fragment, multiple domainbiotherapeutics (e.g., antibody drug conjugates), or related species)that is modified by the term “labeled” includes any entity that isconjugated with another molecule or chemical entity a that isempirically detectable (e.g., “detectable label”). Chemical speciessuitable as labels for labeled-entities include, but are not limited to,enzymes, fluorescent dyes; quantum dots; optical dyes; luminescent dyes;and radionuclides.

As used herein, the term “one or more” includes at least one, moresuitably, one, two, three, four, five, ten, twenty, fifty, one-hundred,five-hundred, etc., of the item to which “one or more” refers.

The approach disclosed herein has been shown to eliminate druginterference in ADA assays. In practice, this method principle can beapplied to reduce/eliminate the interferences in any type ofimmunoassay. This method principle can also be used for any ligandbinding assays for ADA, PK and biomarkers. The methods described hereincan be applied to ligand binding assays to test for neutralizingantibodies (NAbs). The ligand binding assays can include competitiveinhibition of drug binding to drug target. In PK and biomarker assays,excess antibody can be added for complex formation and afterprecipitation and acid dissociation; detection is made using labeleddetection antibody. In all cases, it is important to optimize theconcentration of PEG in the assay to balance the sensitivity andspecificity. The higher concentration of PEG, the lower molecular weightprotein it will precipitate. Therefore, to specifically precipitatedesired complex containing target analyte (such as antibody-drug complexprecipitation), one needs to minimize the amount on unbound non-specificproteins to be precipitated (such as serum IgM and IgG). The MSD highbind plate was utilized in the studies due to its carbon and porousstructure. Based on assay design principle, other large capacity coatingsurfaces would also work.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic representation of an embodiment of an ADA assayaccording to the invention.

FIGS. 2A and 2B are graphs depicting the results of an example bridgingassay format detecting affinity purified rabbit antibody at levelsranging from 8 μg/mL to 125 ng/mL with various concentration of Drug A(0, 1, 10 and 100 μg/mL) tested in the MSD bridging assay format withoutacid dissociation. A. The observed S/B was plotted against the ADAconcentration to assess the recovery of the antibody with differentlevels of drug compared to baseline without drug. B. Percent recoveryrelative to baseline. ADA: MSD bridging assay: Meso Scale Discovery®bridging; S/B: Signal-to-background.

FIGS. 3A and 3B are graphs depicting the results of an example bridgingassay format detecting affinity purified rabbit antibody at levelsranging from 8 μg/mL to 125 ng/mL with various concentration of Drug A(0, 1, 10 and 100 μg/mL) tested in the MSD bridging assay format withacid dissociation. A. The observed S/B was plotted against the ADAconcentration to assess the recovery of the antibody with differentlevels of drug compared to baseline without drug. B. Percent recoveryrelative to baseline. S/B: Signal-to-background.

FIGS. 4A and 4B are graphs depicting the results of an example bridgingassay format detecting affinity purified rabbit antibody at levelsranging from 8 μg/mL to 125 ng/mL with various concentration of Drug A(0, 1, 10 and 100 μg/mL) tested in the MSD bridging assay using thepolyethylene glycol precipitation and acid dissociation assay format ofthe invention (i.e., the PandA assay format). A. The observed S/B wasplotted against the ADA concentration to assess the recovery of theantibody with different levels of drug compared to baseline withoutdrug. B. Percent recovery relative to baseline. S/B:Signal-to-background.

FIG. 5 is a graph depicting the results of an example bridging assayformat detecting affinity purified rabbit anti-Drug A antibody with nodrug tested in the PEG and Acid (PandA) assay format using threedifferent lots of MSD high bind plates. The observed S/B was plottedagainst the ADA concentration. ADA: Anti-drug antibody; S/B:Signal-to-background.

FIG. 6 is a graph depicting the results of an example bridging assayformat detecting Drug B ADAs in pooled normal serum samples (n=32)evaluated in the MSDB assay format with and without acid dissociation.Normal Population Distribution (Drug B bridging assays). Notreatment=without dissociation; Acid Treated=with acid dissociation;S/B: Signal-to-background.

FIG. 7 is a graph depicting the results of an example bridging assayformat detecting Drug B ADAs in pooled disease baseline serum samples(n=16) evaluated in the MSD bridging assay format with and without aciddissociation, as well as the PandA assay format to determine populationdistribution.

FIGS. 8A and 8B are graphs depicting the results of an example bridgingassay format detecting affinity purified rabbit antibody at levelsranging from 4 μg/mL to 31.3 ng/mL with various concentration of Drug B(0, 10, 50 and 250 μg/mL) tested in the MSD bridging assay formatwithout acid dissociation. A. The observed S/B was plotted against theADA concentration to assess the recovery of the antibody with differentlevels of drug compared to baseline without drug. B. Percent recoveryrelative to baseline. S/B: Signal-to-background.

FIGS. 9A and 9B are graphs depicting the results of an example bridgingassay format detecting affinity purified rabbit antibody at levelsranging from 4 μg/mL to 31.3 ng/mL with various concentration of drug B(0, 10, 50 and 250 μg/mL) tested in the MSD bridging assay using thePandA assay format. A. The observed S/B was plotted against the ADAconcentration to assess the recovery of the antibody with differentlevels of drug compared to baseline without drug. B. Percent recoveryrelative to baseline. S/B: Signal-to-background.

FIGS. 10A and 10B are graphs depicting the results of an examplebridging assay format detecting affinity purified rabbit antibody atlevels ranging from 8 μg/mL to 125 ng/mL with various concentration ofDrug C (0, 2.5, 25 and 250 μg/mL) tested in the MSD bridging assayformat with acid dissociation. A. The observed S/B was plotted againstthe ADA concentration to assess the recovery of the antibody withdifferent levels of drug compared to baseline without drug. B. Percentrecovery relative to baseline. S/B: Signal-to-background.

FIGS. 11A and 11B are graphs depicting the results of an examplebridging assay format detecting affinity purified rabbit antibody atlevels ranging from 8 μg/mL to 125 ng/mL with various concentration ofDrug C (0, 2.5, 25 and 250 μg/mL) evaluated in the MSD bridging assayusing the PandA assay format. A. The observed S/B was plotted againstthe ADA concentration to assess the recovery of the antibody withdifferent levels of drug compared to baseline without drug. B. Percentrecovery relative to baseline. S/B: Signal-to-background.

DETAILED DESCRIPTION

The present inventors have developed a novel approach for qualitativelyand/or quantitatively detecting ADAs from a sample which is effective inreducing and eliminating the interference problems caused by drug ortarget in ADA detection. Using the principle of PEG precipitation, aciddissociation, coating on high capacity surface under acidic condition,the methods described herein allow for specific detection of ADA as wellas drug or drug target using specific detection reagent. The approachcan be applied to broader applications for reduction or elimination ofinterference in immunoassays for ADA, PK/TK, and biomarker (such as drugtarget) assays, as well as ligand-binding assays for detection ofneutralizing antibodies.

For a drug with a long half-life and/or one administered at a high doseor a repeated dose, such as an antibody-based therapy, the ADA usuallyforms circulating immune complexes with the drug, typically making theADA unavailable for detection. For example, circulating drug mayinterfere with the detection of ADAs and drug target, or ADAs mayinterfere with the detection and/or accurate quantitation of drug levelsfor pharmacokinetic (“PK”) studies and or toxicokinetics (“TK”) studies.Monoclonal antibody drug interference, especially from human IgG4 drugs,presents an additional challenge for ADA analysis due to its longerhalf-life and higher doses. The impact of such interference is specificto the immunoassay method and platform and may be dependent on thereagents used in each respective assay. Development of drug tolerantimmunogenicity assays becomes more challenging when the drug itself is ahumanized antibody therapeutic.

The most widely adopted approaches in use currently still havelimitations of timing, sensitivity or accuracy due to the presence ofinterference factors that are the same or resemble the binding partnersin the ligand assay. These interferences include but not limited to druginterference in the ADA assay, ADA and/or drug target interference inthe PK assay, drug interference in the drug target biomarker assay, etc.The commonly used ADA method is the bridging assay where a multi-valentADA bridges between a capture drug (unlabeled or biotin labeled) and alabeled detection drug. This format is susceptible to endogenous druginterference (false negative ADA) and/or drug target interference (falsepositive ADA). Being recognized as one of the major challenges in theanalytical method development field, many approaches have been used tomitigate this problem such as acid or base dissociation, 3rd partybinding partner competitive inhibition, and/or removal of theinterference factors, solid phase extraction (SPEAD), ACE and manyothers. The use of acid dissociation in sample treatment in conjunctionwith a bridging assay allows for some improvement in the detection ofADAs in the presence of a drug with improved assay sensitivity and ADArecovery. The same is typically observed with the SPEAD and ACE whichusually allow for some improvement in drug tolerance; but sensitivityand relative accuracy is not fully maintained. Although acid (or base)dissociate the ADA/drug immune complex, once the mixture is neutralizedunder the assay condition, the immune complexes re-form. In currentlyused ADA assays, ADA detection is only possible if the production of ADAexceeds the amount of drug present in the patient's serum due to theformation of ADA-drug complexes. Such drug interference leads to anunderestimation of the number of patients of patients producing ADA.

Immunogenicity of drug products, particularly therapeutic proteins, is amajor concern in clinical and preclinical studies since it can lead topotentially serious side effects, loss of efficacy, and changes in drugexposure, complicating the interpretation of toxicity, pharmacokinetic(PK) and pharmacodynamics (PD) data. As the number of drug products withlong half-life such as monoclonal antibodies is increasing, drugtolerance in ADA assays is of growing concern. To assess the overallimmunogenic potential of a drug, the total amount of ADA is ofparticular interest. Many techniques that are widely used for detectionof ADAs in serum and plasma depend on the specific binding of the ADA toits target drug via the antigen binding site. For example, bridgingassay formats rely on the availability of the antigen binding sites onADAs. As these many assay formats rely on the availability of theantigen binding sites on the ADAs, only drug-free or partially drug-freeADA can be detected. Because specific assay materials are sparse andtime is pressing, an assay format with improved drug tolerance fordetection of ADAs in biological samples is highly desirable

Drug tolerance is generally defined as the maximal amount of free drugin a sample that still results in a detectable ADA signal. Acidtreatment of samples has been used to improve free drug tolerance in ADAassays. Antibody-antigen (or drug) binding is weakened and eventuallydisrupted by low pH, making detection of free ADA that is dissociatedfrom partially or completely drug-bound ADAs possible in manyimmunogenic assay formats (i.e., bridging assay formats), therebyimproving drug tolerance. As demonstrated in the examples providedherein, however, acid treatment alone does not eliminate drug tolerancein ADA assays.

In some embodiments, antibody-antigen (or drug) binding is weakened andeventually disrupted by high pH, making detection of free ADA that isdissociated from partially or completely drub-bound ADAs, followingtreatment with a basic solution, possible in many immunogenic assayformats. The structural characteristics of the biologic drug, such aspI, or the presence of certain conjugating bonds, will dictate whetheran acid solution or basic solution is more appropriate for disruptingADA/drug binding. To increase drug tolerance and provide an improved ADAassay format, the present inventors have developed methods fordetermining the presence or absence of an ADA in a sample with improveddrug tolerance. The methods contemplated herein include a polyethyleneglycol precipitation step and an acid dissociation step.

As disclosed herein, the present invention relates to a method fordetermining the presence or absence of an ADA in a sample (e.g., abiological sample), the method comprising contacting the sample with anexcess amount of drug to which the ADA binds to form drug/ADA complexes,contacting the drug/ADA complexes with polyethylene glycol (PEG), toform a precipitate comprising drug/ADA complexes, contacting theprecipitate with a solution to dissociate the drug/ADA complexes,immobilizing the dissociated ADAs on a substrate, and determining thepresence of or amount of said ADA. In a further aspect, the determiningstep comprises contacting the immobilized ADAs with drug conjugated to adetectable label and determining the presence of or amount of saiddetectable label, to thereby determine the presence or absence of ADA inthe sample.

In one embodiment, the precipitate (drug/ADA complexes) is contactedwith an acid solution to dissociate the complexes and then coated on alarge surface (under acidic conditions to keep drug and ADA apart) orsubstrate (e.g., a high bind carbon plate with high coating capacity)for a time sufficient to allow coating of all dissociated free drugs andfree ADAs. The acidic environment prevents the complexes from reformingwhile being immobilized onto the substrate surface. When an acidsolution is used to dissociate the complexes, the assay can be referredto as a PandA (PEG and Acid) assay.

In another embodiment, the final precipitate (drug/ADA complexes) isreconstituted with a basic solution to dissociate the complexes and thencoated on a large surface (under basic conditions to keep drug and ADAapart) or substrate (e.g., a high bind carbon plate with high coatingcapacity) for a time sufficient to allow coating of all dissociated freedrugs and free ADAs. The basic environment prevents the complexes fromreforming while being immobilized onto the substrate surface.

The selection of an acidic or basic solution will depend on theparameters of the drug (e.g., the biologic drug), such as pI, or thepresence of certain conjugating bonds, and the selection will haveminimal effect on the integrity and structure of the drug.

The present invention also relates to methods for reducing interferencein a drug PK/TK assay due to the presence of an ADA in a biologicalsample, the method comprising contacting the sample with an excessamount of an ADA to form drug/ADA complexes, contacting the drug/ADAcomplexes with polyethylene glycol (PEG), to thereby form a precipitatecomprising drug/ADA complexes, contacting the precipitate with an acidsolution, thereby dissociating the drug/ADA complexes, immobilizing thedissociated free drug and free ADA on a surface and/or substrate, andperforming the drug PK/TK assay using specific detection reagent againstdrug, to thereby reduce interference from the ADA. The drug assay canbe, for example, a drug quantitation assay, a drug PK/TK assay or a drugpotency assay. Similar principle can be applied to method forquantitation of drug target as biomarker assay for drug safety andefficacy (PD) in the presence of drug interference. Excess drug is addedto the sample to form drug target/drug complex. PEG precipitation andacid dissociation (or base dissociation) steps are used and specificdrug target detection reagent is then used.

In some aspects the disclosure provides methods for determining thepresence or absence of an ADA directed against a drug. The term “drug”or “drug material”, as used herein refers to a chemical that hasmedicinal, performance-enhancing, and/or intoxicating effects whenintroduced into the body of a human or other animal. For example, thedrug can be an organic or inorganic small molecule compound or abiologic therapeutic (e.g., an antibody (e.g., a drug antibody) orfragment thereof, multiple domain biotherapeutics, nucleic acid,peptide, polypeptide, peptidomimetic, carbohydrate, or lipid), so longas the drug is immunogenic and capable of eliciting an immune response.The term “drug antibody” denotes an antibody which can be administeredto an individual for the treatment of a disease and as used hereindistinguishes such antibodies from ADAs. Non-limiting examples of drugantibodies include, for example, an antibody selected frommuromomab-CD3, abciximab, rituximab, daclizumab, basiliximab,palivizumab, infliximab, trastuzumab, etanercept, gemtuzumab,fresolimumab, alemtuzumab, ibritomomab, adalimumab, alefacept,omalizumab, tofacitinib, tositumomab, efalizumab, cetuximab,bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumabmepolizumab, necitumumab, blinatumomab, nivolumab, dinutuximab,secukinumab, evolocumab, pembrolizumab, ramucirumab, vedoluzumab,siltuximab, opinutuzumab, adotrastuzumab emtansine, raxibacumab,pertuzumab, brentuximab, belimumab, ipilimumab, denosumab, tocilizumab,ofatumumab, canakinumab, golimumab, ustekinumab, catumaxomab, andcertolizumab.

The methods disclosed herein may comprise a polyethylene glycol (“PEG”)mediated precipitation step comprising contacting a sample with a PEGcompound. The PEG compound may be a PEG compound having a molecularweight between 1,000 and 40,000 daltons. For example, the PEG compoundcomprises at least one PEG selected from the group consisting ofselected from the group consisting of PEG1000, PEG1450, PEG3000,PEG6000, PEG8000, PEG10000, PEG14000, PEG15000, PEG20000, PEG250000,PEG30000, PEG35000, and PEG40000.

The specific concentration of PEG is selected to maximize the balance ofspecificity and selectivity. The amount of PEG contacted with the samplemay correspond to a concentration of between about 0.1% and about 10.0%,about 0.2% and about 7.0%, between about 0.5% and about 6.0%, betweenabout 0.5% and about 5.0%, between about 1.5% and about 5.5%, betweenabout 2.0% and about 5.0%, between about 3.0% and about 4.5%, betweenabout 3.5% and about 4.0%, between about 1.0% and about 2.5%, betweenabout 1.2% and about 1.5%, or about 0.1%, 0.2%, 0.5%, 1.0%, 1.2%, 1.5%,2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%,8.0%, 8.5%, 9.0%, 9.5% or about 10.0% PEG.

The methods may comprise an acid dissociation step comprising contactinga precipitate with an acid. The acid may be or include an organic acid.Alternatively or in addition, the acid may be or include an inorganicacid. The acid used in the dissociation step may comprise a mixture ofan organic acid and an inorganic acid. Non-limiting examples of organicacids include, for example, citric acid, isocitric acid, glutamic acid,acetic acid, lactic acid, formic acid, oxalic acid, uric acid,trifluoroacetic acid, benzene sulfonic acid, aminomethanesulfonic acid,camphor-10-sulfonic acid, chloroacetic acid, bromoacetic acid,iodoacetic acid, propanoic acid, butanoic acid, glyceric acid, succinicacid, malic acid, aspartic acid, and combinations thereof. Non-limitingexamples of inorganic acids include, for example, hydrochloric acid,nitric acid, phosphoric acid, sulfuric acid, boric acid, hydrofluoricacid, hydrobromic acid, and mixtures thereof.

The amount of an acid may correspond to a concentration of between about0.01M to about 10M, between about 0.1M to about 5M, about 0.1M to about2M, between about 0.2M to about 1M, or between about 0.25M to about0.75M of an acid or a mixture of acids. In some instances the amount ofan acid corresponds to a concentration of greater than or equal to about0.01M, 0.05M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M,2M, 3M, 4M, 5M, 6M, 7M, 8M, 9M, or 10M of an acid or a mixture of acids.The pH of the acid can be, for example, about 0.1, 0.5, 1.0, 1.5, 2.0,2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or 6.5.

The methods may comprise an base dissociation step comprising contactinga precipitate with an base. The base may be or include an organic base.Alternatively or in addition, the acid may be or include an inorganicbase. The base used in the dissociation step may comprise a mixture ofan organic base and an inorganic base. Non-limiting examples of basesinclude, for example, urea, sodium hydroxide, rubidium hydroxide, cesiumhydroxide, calcium hydroxide, strontium hydroxide, barium hydroxide,zinc hydroxide, lithium hydroxide, acetone, methylamine, and ammonia,and mixtures thereof.

Where a basic solution is used to disrupt the ADA/drug interaction, theamount of base may correspond to a concentration of between about 0.01Mto about 5M, between about 0.1M to about 5M, about 0.1M to about 1M,between about 0.2M to about 1M, or between about 0.25M to about 0.75M ofa base or a mixture of bases. In some instances the amount of a basecorresponds to a concentration of greater than or equal to about 0.01M,0.05M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 2M, 3M,4M, 5M, 6M, 7M, 8M, 9M, or 10M of a base or a mixture of bases. The pHof the base can be, for example, about 8.0, 8.5, 9.0, 9.5, 10.0, 10.5,11.0, 11.5, 12.0, 12.5 and 13.0.

In some embodiments, the sample is contacted with an acid or base for anamount of time sufficient to dissociate preformed drug/ADA complexes. Incertain instances, the sample is contacted (e.g., incubated) with anacid or base for a period of time ranging from about 0.1 hours to about24 hours, e.g., about 0.2 hours to about 16 hours, about 0.5 hours toabout 10 hours, about 0.5 hours to about 5 hours, or about 0.5 hours toabout 2 hours. In other instances, the sample is contacted (e.g.,incubated) with an acid or base for a period of time that is greaterthan or equal to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 hours. The sample canbe contacted with an acid or a base at any temperature that is generallycompatible with the method, e.g., 4° C., room temperature (RT), or 37°C. RT can be, for example 22° C. to 26° C., e.g., 23° C., 24° C. or 25°C.

The methods may comprise immobilizing a dissociated ADA and/or drug on asurface and/or substrate. The substrate may comprise a carbon surface,glass surface, silica surface, metal surface, a surface coated with apolymeric material, a surface containing a metallic or chemical coating,a membrane, micro-beads, or a porous polymer matrix. The substrate maycomprise a porous carbon surface. In an exemplary embodiment, thesubstrate is a high bind carbon plate having a large surface and highcoating capacity.

The methods may further comprise determining the presence of or amountof an ADA, or the presence of or amount of a drug in the sample. Thus,the disclosure provides antibodies, anti-drug antibodies or drug labeledwith a detectable label. Non-limiting examples of detectable labels forany of the methods of the invention include a hapten, an enzyme, anenzyme substrate, an enzyme inhibitors, a fluorophore, a chromophores,luminescent markers, radioisotopes (including radionucleotides) and amember of a binding pair. The intensity of the detectable label can bemeasured using instruments and devices known to those skilled in theart, including, for example a portable or benchtop fluorometer (e.g., ahandheld fluorometer) or a portable or benchtop colorimeter (e.g., ahandheld colorimeter).

In some embodiments, the detectable label is an electrochemiluminescentlabel, including, for example, a Sulfo-TAG® label.

The detectable label can be a specific member (a first member or asecond member) of a binding pair. Binding pairs for use in the methodsprovided herein include, for example, biotin/streptavidin,biotin/avidin, biotin/neutravidin, biotin/captavidin, epitope/antibody,protein A/immunoglobulin, protein G/immunoglobulin, proteinL/immunoglobulin, GST/glutathione, His-tag/Nickel, antigen/antibody,FLAG/M1 antibody, maltose binding protein/maltose, calmodulin bindingprotein/calmodulin, enzyme-enzyme substrate, and receptor-ligand bindingpairs. In some embodiments, the GlcNac binding protein is conjugated toa first member of binding pair (e.g., biotin, avidin, neutravidn,captavid, antibody, antigen, protein A, protein G, protein L, GST,His-Tag, FLAG, MBP, calmodulin binding protein, an enzyme, a receptor orligand).

As used herein, the terms “fluorescence label” and “fluorophore” usedinterchangeably and refer to any substance that emits electromagneticenergy such as light at a certain wavelength (emission wavelength) whenthe substance is illuminated by radiation of a different wavelength(excitation wavelength) and is intended to encompass a chemical orbiochemical molecule or fragments thereof that is capable of interactingor reacting specifically with an analyte of interest in a sample toprovide one or more optical signals.

Representative fluorophores for use in the methods provided hereininclude, for example, green fluorescent protein, blue fluorescentprotein, red fluorescent protein, fluorescein, fluorescein5-isothiocyanate (FITC), cyanine dyes (Cy3, Cy3.5, Cy5, Cy5.5, Cy7),Bodipy dyes (Invitrogen) and/or Alexa Fluor dyes (Invitrogen), dansyl,Dansyl Chloride (DNS-C1), 5-(iodoacetamida)fluorescein (5-IAF,6-acryloyl-2-dimethylaminonaphthalene (acrylodan),7-nitrobenzo-2-oxa-1,3-diazol-4-yl chloride (NBD-Cl), ethidium bromide,Lucifer Yellow, rhodamine dyes (5-carboxyrhodamine 6G hydrochloride,Lissamine rhodamine B sulfonyl chloride, rhodamine-B-isothiocyanate(RITC (rhodamine-B-isothiocyanate), rhodamine 800); tetramethylrhodamine5-(and 6-) isothiocyanate (TRITC)), Texas Red™, sulfonyl chloride,naphthalamine sulfonic acids including but not limited to1-anilinonaphthalene-8-sulfonic acid (ANS) and6-(p-toluidinyl)naphthalen-e-2-sulfonic acid (TNS), Anthroyl fatty acid,DPH, Parinaric acid, TMA-DPH, Fluorenyl fatty acid,Fluorescein-phosphatidylethanolamine, Texasred-phosphatidylethanolamine, Pyrenyl-phophatidylcholine,Fluorenyl-phosphotidylcholine, Merocyanine 540, Naphtyl Styryl,3,3′dipropylthiadicarbocyanine (diS-C3-(5)), 4-(p-dipentylaminostyryl)-1-methylpyridinium (di-5-ASP), Cy-3 lodo Acetamide,Cy-5-N-Hydroxysuccinimide, Cy-7-Isothiocyanate, IR-125, Thiazole Orange,Azure B, Nile Blue, Al Phthalocyanine, Oxaxine1,4′,6-diamidino-2-phenylindole. (DAPI), Hoechst 33342, TOTO, AcridineOrange, Ethidium Homodimer, N(ethoxycarbonylmethyl)-6-methoxyquinolinium(MQAE), Fura-2, Calcium Green, Carboxy SNARF-6, BAPTA, coumarin,phytofiuors, Coronene, and metal-ligand complexes.

Haptens for use in the methods provided herein include, for example,digoxigenin, and biotin.

Enzymes for use in the methods provided herein include, for example,alkaline phosphatase (AP), beta-galactosidase, horse radish peroxidase(HRP), soy bean peroxidase (SBP), urease, beta-lactamase and glucoseoxidase.

In certain aspects, this disclosure provides kits that are adapted fordetermining the presence or absence of an ADA in a biological sample.The kits may comprise instructions and, in a container, reagents forcontacting drug/ADA complexes with polyethylene glycol (PEG), to form aprecipitate comprising drug/ADA complexes and reagents for contactingthe precipitate with an acid solution to dissociating the drug/ADAcomplexes; and a substrate suitable for immobilizing the dissociatedADAs or drug on for further analysis.

Examples

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

The examples below describe novel ADA assay methods where completerecovery of an antibody was obtained at the limit of quantitationdespite the presence of high levels of the antibody therapeutic.Specifically, three case studies are provided to demonstrate eliminationof drug interference in ADA assays for the monoclonal antibodytherapeutics (drugs A, B and C).

1.1 Assay Format

To improve the drug tolerance (or eliminate drug interference) ofconventional immunogenicity assays, the inventors sought to develop anew method for determining the presence or amount of an ADA in a sample(e.g., a biological sample). The inventors have successfully developed anew method where complete recovery of anti-drug antibody was obtained atthe limit of quantitation despite the presence of high levels of drug(the antibody therapeutic). A schematic representation of an exemplaryembodiment of the method is shown in FIG. 1.

As depicted in FIG. 1, excess drug material is added to a sample (e.g.,a biological sample) to allow drug/ADA complexes to form. Following theinitial incubation, PEG is added to each sample and incubated to allowfor precipitation of complexes. After a series (e.g., one or more) ofwashes, the precipitate is reconstituted with an acid solution todissociate drug/ADA complexes. The dissociated drug/ADA complexes arecoated on a substrate (i.e., coated on the ells of a high bind MSDplate). Following incubation, the substrate is blocked and thendetection is performed using drug labeled with a detectable labelallowing for ADA detection by ECL (“electrochemiluminescence) or“enhanced chemiluminescence”).

In one aspect, the method depicted in FIG. 1 comprises adding excessdrug material to the samples to saturate free antibody therefore formingdrug/ADA complexes. The complexes are then precipitated using PEG. PEGis added to the sample at a concentration optimized to achieve thedesired sensitivity while maintaining specificity. After a series ofwashes, the final precipitate is reconstituted with an acid solution andcoated on a high bind carbon plate. The acidic environment prevents thecomplexes from reforming while being absorbed onto the porous carbonsurface of the MSD high bind plate. Detection of the total ADA levels isthen performed using Sulfo-TAG® label conjugated drug followed byElectrochemiluminescence read out on a Meso Scale Discovery® Sector 2400reader.

1.2 Experimental Materials

Therapeutic monoclonal antibodies, Sulfo-TAG® drug, and affinitypurified rabbit anti-drug are developed by Genzyme, a Sanofi Company(Framingham, Mass.). Naive human serum pools from healthy individualswere purchased from Bioreclamation Inc. Disease baseline serum sampleswere obtained from treatment naïve subjects enrolled in product-relatedclinical trials. High Bind 96-well Meso Scale plates, Sulfo Tag, readBuffer T, Sector 2400 reader attained from Meso Scale Discovery®(“MSD”). PEG8000 was obtained from TekNova. Glacial acetic acid wasprovided by J. T Baker. Tween 20 and Non-Fat Dry Milk-Sigma, acquiredfrom Aldrich. The ELx405 plate washer was supplied by Biotek. Plate washbuffer came from PerkinElmer. Clear polypropylene 96-well microtiterplates were procured from Corning. Bovine Serum Albumin was obtainedfrom Seracare. Borate is found through Sigma Aldrich, Catalog numberB0394.

Drug A is a humanized IgG1 depleting antibody that binds to lymphocytecell surface target for an autoimmune disease. Drug B is a full humanIgG4 that neutralizes a soluble cytokine binding to its cell surfacereceptor in the target tissue for a fibrosis indication. Drug C is ahumanized IgG4 that recognizes cell surface adhesion molecule and blocksits binding to a soluble ligand.

1.3 Bridging Immunoassay without Acid Dissociation Procedure

The MSD bridging assay format requires the drug to be labeled withbiotin and labeled with sulfo-TAG®. According to the MSD assay format,the biotinylated drug will serve as the capture molecule and thesulfo-TAG® labeled drug will be the reporter in the bridging assay.

Samples are initially diluted 1:10 in assay buffer ((300 mM Acetic acid,2% BSA). Samples are added to a polypropylene plate in duplicate wellsand a solution containing equi-molar concentrations of Biotin-Drug andSulfo-TAG-Drug in assay buffer is added. The plate is then incubated for2 hours in a 22-26° C. shaker at 450 rpm. A streptavidin coated MSDplate is blocked with assay buffer for a minimum of one hour. Followingthe incubation, the MSD plate is washed 3 times and 50 μL of theincubated mixture is transferred from the polypropylene plate to the MSDplate and incubated for 2 hours on a 22-26° C. shaker. Following theincubation, the plate is washed and a solution of MSD read buffer Tcontaining tripropylamine is added and the plate is read on the MSDsector imager 2400.

Within the instrument, a voltage is applied. In the presence oftripropylamine, sulfo-TAG® participates in an electro-chemiluminescent(ECL) reaction. The antibodies bridging the sulfo-TAG-Drug and theBiotin-Drug bound to the streptavidin surface will result in an ECLsignal. After the final incubation, the plate is washed with 0.05% Tweenin PBS. Read buffer T 2× is then added and the plate is read on a SectorPR2400. The electro-chemiluminescent signal is proportional to theanti-drug antibody in each sample. Sample results are converted to asignal to background ratio (S/B) by dividing the average ECL signal froman individual sample by the average ECL signal of the negative control.

1.4 Bridging Immunoassay with Acid Dissociation Procedure

Samples are initially diluted 1:10 in assay buffer (300 mM Acetic acid,2% BSA) in incubated at 22-26° C. for 45 minutes. Samples are then addedto a polypropylene plate in duplicate wells and a solution containingequi-molar concentrations of Biotin-Drug and sulfo-TAG-Drug in assaybuffer in addition to Tris HCL is added at a ratio to effectivelyneutralize the pH to 7.0. The plate is then incubated for 2 hours in a22-26° C. shaker at 450 rpm. A streptavidin coated MSD plate is blockedwith assay buffer for a minimum of one hour. Following the incubation,the MSD plate is washed 3 times and 50 μL of the incubated mixture istransferred from the polypropylene plate to the MSD plate and incubatedfor 2 hours on a 22-26° C. shaker. Following the incubation, the plateis washed and a solution of MSD read buffer T containing tripropylamineis added and the plate is read on the MSD sector imager 2400.

Within the instrument, a voltage is applied. In the presence oftripropylamine, sulfo-TAG® participates in an electro-chemiluminescent(ECL) reaction. The antibodies bridging the sulfo-TAG-Drug and theBiotin-Drug bound to the streptavidin surface will result in an ECLsignal. After the final incubation, the plate is washed with 0.05% Tweenin PBS. Read buffer T 2× is then added and the plate is read on a SectorPR2400. The electro-chemiluminescent signal is proportional to theanti-drug antibody in each sample. Sample results are converted to asignal to background ratio (S/B) by dividing the average ECL signal froman individual sample by the average ECL signal of the negative control.

1.5 PEG and Acid (PandA) Procedure 1

Samples are initially diluted ⅕ in assay buffer (300 mM Acetic acid, 2%BSA) containing excess drug (10-50 μg/mL) and incubated for one hour at37° C. with 450 rpm in a polypropylene plate to allow complexes betweendrug and any remaining free antibody in the sample. This is followed bythe addition of 3% PEG in Borate pH 8.0 to each sample and an overnightincubation at 2-8° C. The final concentration of PEG buffer in eachsample is 1.5%.

The following day, the plate is centrifuged at 4000 rpms for 20 minutesto precipitate the complexes into a pellet. The pellets are thenre-suspended with 1.5% PEG in Borate pH 8.0 and centrifuged a secondtime at 4000 rpms for 20 minutes. The wash cycle is repeated threetimes. Following the final centrifugation, each sample suspended in 100μl of 300 mM acetic acid and further diluted 1/10 (20 μl sample+180 μlacetic acid) for a final sample dilution of 1/50. Diluted samples arethen coated by adding 25 μl in duplicate to wells of an MSD high bindplate and incubated for one hour at 24° C. shaking at 450 rpm. Followingthe incubation, the plate is washed with 1× plate wash buffer andblocked with 3% milk in PBS for one hour at 24° C. shaking; after which,the plate is washed and 100 ng/ml of sulfo-TAG-Drug was added to thesamples and incubated for one hour at 24° C. shaking. After the finalincubation, the plate is washed with 0.05% Tween in PBS. Read buffer T2× is then added and the plate is read on a Sector PR2400. Theelectrochemiluminescent signal is proportional to the anti-drug antibodyin each sample.

In some embodiments, the incubation step following the initial acidaddition can be carried out at 23° C., 25° C., 27° C., 30° C., 32° C.,35° C., 37° C., 39° C. or higher.

In some embodiments, following the final precipitation step, each samplecan be further diluted to a final sample dilution of, e.g., 1:5, 1:10,1:20, 1:25, 1:30, 1:40, 1:50, or 1:60, 1:80, 1:100, or 1:200. Typicallythe final sample dilution will be <1:100, which is the minimal requireddilution (MRD) set by the Food and Drug Administration (FDA).

1.5 PEG and Acid (PandA) Procedure 2

Samples are initially diluted ⅕ in assay buffer (300 mM Acetic acid, 2%BSA) containing excess drug (10-50 μg/mL) and incubated for one hour at24° C. with 450 rpm in a polypropylene plate to allow complexes to formbetween drug and any remaining free antibody in the sample. This isfollowed by the addition of a PEG solution at a 1:1 PEG:sample ratio toeach sample and an overnight incubation at 2-8° C. The finalconcentration of PEG was optimized for each product to be optimal forprecipitation of specific complexes and achieving the desiredspecificity and sensitivity while minimizing the effect of un-complexedmolecules such as non-specific IgGs. The PEG concentrations added to thesamples were between 3% and 6% (3% for drug A and 6% for drug B and Cadded at a 1:1 ratio to diluted samples).

The following day, the plate is centrifuged at 4000 rpms for 30 minutesto precipitate the complexes into a pellet. The pellets are thenre-suspended with PEG in PBS and centrifuged a second time at 4000 rpmsfor 20 minutes. The wash cycle is repeated one additional time.Following the final centrifugation, each sample is re-suspended anddiluted in 300 mM acetic acid to achieve the final MRD desired for theparticular assay ( 1/50 for Drug A and C and 1/25 for drug B). Samplesare then coated in duplicate wells of an MSD high bind plate. The plateis then incubated for one hour at 24° C. shaking at 450 rpm. Followingthe incubation, the plate is washed with 1× plate wash buffer andblocked with 3% milk in PBS for one hour at 24° C. shaking. The plate isthen washed and a solution containing sulfo-TAG-Drug is added to thesamples and incubated for one hour at 24° C. shaking. After the finalincubation, the plate is washed with 1× plate wash buffer. Read buffer T2× is then added and the plate is read on a Sector PR2400. Theelectro-chemiluminescent signal is proportional to the anti-drugantibody in each sample.

In some embodiments, the incubation step following the initial acidaddition can be carried out at 23° C., 25° C., 27° C., 30° C., 32° C.,35° C., 37° C., 39° C. or higher.

In some embodiments, following the final precipitation step, each samplecan be further diluted to a final sample dilution of, e.g., 1:5, 1:10,1:20, 1:25, 1:30, 1:40, 1:50, or 1:60, 1:80, 1:100, or 1:200. Typicallythe final sample dilution will be <1:100, which is the minimal requireddilution (MRD) set by the Food and Drug Administration (FDA).

2.1 Drug A

Sensitivity and Drug Tolerance Assessment

For Drug A, the PandA method was compared to the traditional MSDbridging assay with and without acid dissociation to improve drugtolerance. Assay sensitivity and drug Tolerance were determined usingaffinity purified rabbit anti-drug at concentrations ranging from 8μg/mL to 125 ng/mL with and without drug (Drug A) at variousconcentrations (0, 0.1, 10 and 100 μg/mL) in the MSD bridging assayformat with (FIG. 2A-B) and without acid (FIG. 3A-B) dissociation. Thesamples containing ADA and drug were prepared in pooled normal humansera and incubated for at least one hour at 37° C. allowing drug/ADAcomplexes to form prior to assaying.

Cut point was determined by evaluating 40 normal human serum samples,calculating the mean and the standard deviation for the samples andcalculating the 95^(th) percentile factor of 1.645-times the standarddeviation and adding it to the mean as is typically recommended. Fordrug A, the new method was compared to the traditional bridging assaywith and without acid dissociation for drug tolerance.

FIG. 2 is a summary of the data comparing the MSD bridging assay formatwithout acid dissociation. The results indicate a strong dose responsefor ADA detection in the absence of drug and inhibition seen with 1μg/mL of drug. (FIG. 2A) FIG. 2B indicates low recoveries of antibodydetection, approximately 10% at the 125 ng/mL of ADA at the lowestconcentration of drug tested of 1 μg/mL. (FIG. 2B) The assay sensitivitywas reduced from 15 ng/mL in the absence of drug to 342 ng/mL with 1μg/mL of drug. The sensitivity in the presence of 100 μg/mL of drug wasreduced to 5143 ng/mL or 5.1 μg/mL.

FIG. 3 is a summary of the data from the MSD bridging assay format withacid dissociation. The results indicate a similar dose response to thebridging assay without acid for ADA detection in the absence of drug andinhibition seen with 1 μg/mL of drug. (FIG. 3A) The percent recoveriesremained acceptable with 1 μg/mL of drug but reduced to 35% at the 125ng/mL of ADA at the 10 μg/mL of drug which is lower than the drug Cmax.(FIG. 3B) The assay detection sensitivity was maintained for the 1 and10 μg/mL of drug at around 15 ng/mL while found to be 262 ng/mL in thepresence of 100 μg/mL of drug. Although the sensitivity of the assaymeets some proposed guidelines of 250-500 ng/mL in this method, thisfinding is specific to this product and antibody combination and may notbe acceptable for other products or with different antibody controls.

FIGS. 4A and 4B are a summary of the data from the PandA (Procedure 2)precipitation format. The results indicate an acceptable dose responsein the absence of drug and no significant inhibition seen due to drugpresent in the samples. (FIG. 4A) The percent recoveries remainedacceptable mostly between 80-120% regardless of the drug amount presentin the samples when compared to the sample results with no drug as areference. (FIG. 4B) Similarly, the assay detection sensitivity wasmaintained at 9-14 ng/mL despite drug present at 100 μg/mL which is 3-4folds higher than the expected Cmax of Drug A.

Table 1 is a summary of the assay sensitivities for ADA detection atvarious concentrations of Drug A tested in each of the methods tested.The assay sensitivity concentrations were obtained by back fitting theS/B cut point from each antibody curve shown in FIGS. 2A, 3A and 4A.ADA: Anti-drug antibody; S/B: Signal-to-background.

TABLE 1 Assay Sensitivity ng/mL Bridging Assay Bridging Assay Drugwithout Acid with Acid PandA (μg/mL) Dissociation Dissociation method 015 15 10 1 342 8 13 10 393 16 9 100 5143 262 14As shown in Table 1, the PandA method not only improved the drugtolerance but also maintained the assay sensitivity at 9-14 ng/mLdespite presence of 100 μg/mL of Drug. Antibody detection recoveryremained mostly between 80 and 120% regardless of the amount of drugpresent in the sample, which is superior to the bridging immunoassaywith acid dissociation.Titer Results Comparison

The PandA method can also be used to accurately report antibody titersin the presence of drug. To determine whether end point titers correlatebetween samples that contain antibody alone (0 μg/ml drug) and othersthat contain an equivalent amount of antibody but high drugconcentration (100 μg/mL drug), test samples were analyzed in the methodwhere dilutions for titration were performed following two differenttitration schemes. The first scheme incorporated a titration prior toPEG precipitation and the second scheme incorporated the titration priorto coating on the high bind plates. The end point titers were identicalbetween the no drug and drug containing samples at the same antibodylevel as well as between the titration schemes based on the assay titercut point value (S/B of 1.2). The end point titer data is summarized inTable 2.

TABLE 2 Sample A B DRUG Level μg/mL 100 0 100 0 Titer (diluted pre-PEG)1600 1600 400 400 Titer (diluted post-PEG) 1600 1600 200 4003.1. MSD High Bind Plates (Lot to Lot) and Overall Precision

To determine whether MSD High Bind plates contribute to assayvariability, coating of samples was performed on three different lots.Data was plotted and analyzed for equivalence using ANOVA.

To determine whether MSD High Bind plates contribute to the assayvariability, coating of samples was performed on three different MSDplate lots. The samples contained affinity purified rabbit antibody withvarious concentrations of drug. The observed S/B was plotted against theADA concentration for each of the three lots of plates as shown in FIG.5. The precision between plate lots was found to be acceptable with CVless than 20% (Table 3) and ANOVA showed no significant differencesbetween the three lots with a p-value of 0.1776.

4.1 Drug B

Target Interference in MSD Bridging Assay with Acid Dissociation

For Drug B, a specific challenge was seen in the MSD bridging assay withacid dissociation since the target for Drug B changes from a monomer toa dimer at low pH causing false positive results. The dimerizationeffect is seen in 100% of normal serum samples and disease baselinesamples in the MSD bridging assay with acid dissociation. Thisphenomenon was similar to what was reported by Dai et al. [Dai S,Schantz A, Clements-Egan A, Cannon M, Shankar G: Development of a methodthat eliminates false-positive results due to nerve growth factorinterference in the assessment of fulranumab immunogenicity. AAPS J.16(3), 464-477 (2014)] where it was found that high apparent incidenceof anti-drug antibody (ADA) in phase 1 studies was the result ofdetection of drug target, a homodimer, due to its ability to bridge drugmolecules. Dai et al. found that the acid-dissociation-basedpretreatment of samples used for mitigating drug interferencedramatically increased drug target interference.

To demonstrate the effect of endogenous target dimerization of the drugtarget due to acid dissociation and false positive results in the assay,normal (n=32) and baseline disease (n=16) serum samples were analyzed inthe bridging assay with and without acid dissociation to highlight theeffect of endogenous target dimerization due to acid dissociation andfalse positive results in the assay.

FIG. 6 is a representation of the normal serum samples tested in the MSDbridging assay with and without acid dissociation. A normal distributionat or near the background of the assay is observed (population on theleft) in the non-acid treated set while some positive results wereobserved with S/B greater than 20 when acid dissociation was applied tothe bridging assay.

FIG. 7 is a representation of disease baseline serum samples whenanalyzed in the MSD bridging assay without and with acid dissociation aswell as the PEG and Acid (PandA) method. Results were comparable betweenthe MSD bridging without acid treatment and PandA method while the acidtreatment resulted in higher S/B levels for the majority of the samplestested suggesting interference from drug target due to the dimerizationeffect at low pH.

As shown in FIGS. 6 and 7, for Drug B, the bridging assay with aciddissociation is not a feasible approach in normal or disease populationdue to the dimerization of the drug target at lower pH causing falsepositive results for all samples with results proportional to the amountof endogenous target. For that reason, the assay sensitivity and drugtolerance for Drug B was only compared between the PandA method and theexisting MSD bridging assay without acid dissociation.

FIGS. 8A-B and 9A-B are summaries of the data for Drug B comparing thebridging assay format without acid dissociation to the PEG and Acidmethod (PandA).

In FIG. 8A, the MSD bridging assay format resulted in an acceptable doseresponse for ADA detection in the absence of drug and completeinhibition seen with 10 μg/mL and a decrease of sensitivity from 47ng/mL in the absence of drug to 2 μg/mL of antibody with 10 μg/mL ofdrug.

For Drug B, the bridging assay without acid dissociation sensitivity wasvalidated at 50 ng/mL with poor drug tolerance. Levels of drug at 250ng/mL inhibited detection of anti-Drug B antibodies at 250 ng/mL andlevels of drug at 1 μg/mL inhibited detection of the antibody at 500ng/mL.

FIGS. 9A-B are a summary of the data from the PandA precipitationformat. The results indicate an acceptable dose response in the absenceof drug and no inhibition seen due to drug present in the samples. Thepercent recoveries remained acceptable mostly between 80-120% regardlessof the drug amount present in the samples when compared to the sampleresults with no drug as a reference. The assay detection sensitivity wasmaintained at 39 to 63 ng/mL despite drug present at 250 μg/mL, which ishigher than the expected Cmax.

5.1 Drug C

Sensitivity and Drug Tolerance Assessment

For Drug C, the assay sensitivity and drug tolerance was comparedbetween the new method and the existing MSD bridging assay with aciddissociation where expected drug tolerance cannot be achieved.

FIGS. 10A-B and 11A-B are summaries of the data for Drug C comparing thebridging assay format with acid dissociation to the PEG and Acid method(PandA).

In FIGS. 10A-B, the MSD bridging assay format resulted in an acceptablesensitivity in the absence of drug but inhibition was seen with aslittle as 2.5 μg/mL of drug despite acid treatment in the bridgingimmunoassay. The sensitivity of the assay changed from 227 ng/mL to 2788ng/mL in the presence of 25 μg/mL and antibodies were completelyundetectable in the presence of 250 μg/mL of Drug, levels expected insome clinical samples.

FIGS. 11A-B shows the PandA results to be superior to the bridgingimmunoassay with acid dissociation where antibody detection wasmaintained with complete recovery even in the presence of 250 μg/mL ofdrug. Percent recoveries were observed mostly between 80-120% regardlessof the drug amount present in the samples when compared to the sampleresults with no drug as a reference. The assay detection sensitivity wasmaintained between 129-175 ng/mL despite drug present at 250 μg/mL,which is higher than what is expected in clinical samples.

The examples above describe case studies for three humanized monoclonalantibodies A-C (an IgG1 and 2 IgG4 drugs).

The three drug specific PandA ADA assays resulted in complete recoveryof ADA in samples containing drug levels in excess of those expected inpatients, in contrast to the commonly used assay dissociation approachin MSD bridging assays. This breakthrough novel method shows significantimprovement over the current approaches. In fact, the drug interferenceor under detecting of ADA in all three cases were completely eliminatedand this assay principle could be used not only for ADA assays but alsoPK and biomarker (drug target) analysis in the presence of interferencefactors.

The PandA ADA assay method described herein was shown to be effective atimproving both detection and recovery of ADA in samples containinginterferent levels in excess of what is clinically relevant. The methodalso reported consistent antibody titers regardless of the amount ofdrug present. The method was shown to be superior to the traditionalsolution based bridging assay with acid dissociation maintaining the ADAdetection sensitivity at drug levels in excess of expected clinical Cmaxlevels and no significant inhibition.

The PandA method can also be applied to PK assays where an ADA or DrugTarget is an interferent. Simply, excess antibody (anti-Idiotype) orTarget would be added to form complexes and detection will be using alabeled anti-Idiotype that is specific to the drug. In addition, themethod can be applicable in drug target biomarker assays with potentialdrug interference. In summary, the inventors have described a novelapplication for PEG precipitation of complexes that resolves drug andpossibly target interferences in an ADA immunoassay.

Many different methods and platforms have been used with limited successto address circulating drug interference in immunoassays for thedetection of ADA. This disclosure a novel method that employs PEG andAcid (PandA) that eliminates drug and possibly target interferences inan ADA immunoassay. The novel method showed complete elimination of druginterference at high drug concentrations and demonstrated whilemaintaining assay sensitivity in contrast to the traditional solutionbased MSD bridging assay without or even with acid dissociation. Byapplying the following assay components, the PandA assay hasdemonstrated its intended use to sensitively and specifically detect ADAin the presence of drug and/or drug target: addition of excess drugmaterial to form drug/ADA complexes; precipitation using polyethyleneglycol to get total ADA; acid dissociation and coating of reconstitutedprecipitate in an acidic solution on a high bind carbon plate with alarge capacity to allow for binding to dissociated ADA; and specificdetection of the total ADA levels sulfo-TAG® conjugated drug with an ECLoutput.

In addition, this method also reported consistent antibody titersregardless of the amount of drug present in the sample showing assayprecision. Also reported, the method effectively resolve targetinterference causing false positive results due to target dimerizationin MSD bridging immunoassays with acid dissociation.

In summary, the method is superior to the traditional bridging with aciddissociation method as evidenced by the three proof of principle studiesreported above where complete recovery and detection of ADA in sampleswas achieved with high drug amounts in the samples. This method wassuccessfully validated according to current regulatory expectations andclinical samples tested.

The PandA method described here has shown significant improvement forADA detection in the presence of excess drug. It has a broadapplications based on the principles: (1) saturate free analyte to formall bound analyte in a complex and (2) precipitate the complexes (3)acid dissociate to free analyte without neutralization to reduce analytere-bound while coating the free analyte under acidic condition onto alarge coating surface to immobilize free analyte, and (4) detect freeanalyte using specific reagent. In the examples above, the inventorshave provided three immunogenicity case studies to demonstrate theutility of this novel technology.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for determining the presence or absenceof an anti-drug antibody (ADA) in a sample, the method comprising:contacting the sample with an excess amount of drug to which the ADAbinds to saturate the ADA and to form drug/ADA complexes; contacting thedrug/ADA complexes with polyethylene glycol (PEG), to form a precipitatecomprising drug/ADA complexes; contacting the precipitate with a basicsolution to dissociate the drug/ADA complexes, thereby formingdissociated ADAs and dissociated drugs, wherein the basic solutioncauses the solution of dissociated ADAs and dissociated drugs to have aspecific basic pH; immobilizing the dissociated ADAs on a substrateunder conditions where dissociation of the ADAs and the drugs ismaintained, wherein the conditions where the dissociation of the ADAsand the drugs are maintained is that the solution of dissociated ADAsand dissociated drugs is maintained at the specific basic pH; anddetermining if the ADA is present or absent in the sample.
 2. The methodof claim 1, wherein the determining step comprises contacting theimmobilized ADA with drug labeled with a detectable label; anddetermining the presence or absence of said detectable label, to therebydetermine the presence or absence of ADA in the sample.
 3. The method ofclaim 1, wherein the substrate comprises a porous carbon surface.
 4. Themethod of claim 1, wherein the substrate comprises a carbon surface,glass surface, silica surface, metal surface, a polymeric material, asurface containing a metallic or chemical coating, a membrane, a bead, aporous polymer matrix, or substrates comprising cellulosic fibers, orany combination thereof.
 5. The method of claim 2, wherein thedetectable label comprises a label selected from the group consisting ofa radioactive isotope, an enzyme, a fluorescent label, achemiluminescent label, an electrochemiluminescent label, and asubstrate for an enzymatic detection reaction.
 6. The method of claim 1,wherein the drug comprises an antibody or functional fragment thereof,nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid,an organic small molecule compound, an inorganic small moleculecompound, or any combination thereof.
 7. The method of claim 1, whereinthe drug is a drug modified to exhibit less immunogenicity as comparedto the same drug in unmodified form.
 8. The method of claim 1, whereinthe PEG comprises at least one PEG selected from the group consisting ofPEG1000, PEG1450, PEG3000, PEG6000, PEG8000, PEG10000, PEG14000,PEG15000, PEG20000, PEG250000, PEG30000, PEG35000, and PEG40000.
 9. Themethod of claim 1, wherein the sample is contacted with PEG at aconcentration of between about 0.1% and about 10.0%.
 10. The method ofclaim 1, wherein the sample comprises the drug.
 11. The method of claim1, wherein the method further comprises immobilizing the drug on thesubstrate before or after the step of immobilizing the ADA on thesubstrate.
 12. The method of claim 1, wherein the sample comprises abiological sample, wherein the biological sample comprises materialselected from the group consisting of body fluids, mucus secretions,saliva, blood, whole blood, plasma, and serum.
 13. The method of claim1, wherein the basic solution comprises an organic base, an inorganicbase, or a mixture thereof.
 14. The method of claim 1, wherein the basicsolution comprises a base at a concentration of between about 0.1 M toabout 5 M.
 15. The method of claim 1, wherein the basic solutioncomprises a base selected from the group consisting of urea, sodiumhydroxide, rubidium hydroxide, cesium hydroxide, calcium hydroxide,strontium hydroxide, barium hydroxide, zinc hydroxide, lithiumhydroxide, acetone, methylamine, ammonia, and any combination thereof.